Araştırma Makalesi
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Effect of DNA Concentration on Band Intensity and Resolution in Agarose Gel Electrophoresis

Yıl 2021, Cilt: 14 Sayı: 3, 326 - 333, 30.12.2021
https://doi.org/10.52976/vansaglik.969547

Öz

Objective: Agarose gel electrophoresis (AGE) is a widely used method for separating, identifying, and purifying nucleic acids. It has been used intensively in molecular researches and biotechnological applications. In AGE, band resolution and band quality are important parameters for researchers. Until now, many factors such as comb thickness, gel concentration, voltage and buffers have been reported to influence resolution and band quality in AGE. However, effect of DNA concentration on band resolution and intensity in AGE is unclear. Therefore, aim of the study was to investigate the effect of DNA concentration on AGE results in detail.
Materials and Methods: Different concentrations of DNA Marker and specific DNA fragments obtained by PCR were analyzed, but the loaded total DNA quantity was not changed. Furthermore, the effect of DNA concentration on AGE was also investigated by designed gel combs with different thicknesses and wideness.
Findings: It was shown that DNA concentration did not affect gel resolution, and the effect was not changed by comb thickness and wideness. Also, 2-fold dilution did not affect band intensities while 8-fold dilution significantly affected band intensities of all tested DNA fragments.
Conclusion: Concentrating samples, at least 8-fold, and using thin and narrow gel combs can be used for desired results in AGE.

Kaynakça

  • Aaij C, Borst P. The gel electrophoresis of DNA. Biochim Biophys Acta 1972; 269:192-200
  • Arslan M. A new primer designing for PCR-RFLP analysis of A and B genetic variants of bovine kappa-casein. Harran Üniversitesi Veteriner Fakültesi Dergisi 2020; 9:6-11
  • Bachvaroff R, McMaster PR. Separation of microsomal RNA into five bands during agar electrophoresis. Science 1964; 143:1177-1179
  • Greaser ML, Warren CM. Protein electrophoresis in agarose gels for separating high molecular weight proteins. Methods Mol Biol 2012; 869:111-118
  • Green MR, Sambrook J. Analysis of DNA by Agarose Gel Electrophoresis. Cold Spring Harbor Protocols 2019; 2019
  • Hall AC. A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis. bioRxiv 2020; 568253
  • Helling RB, Goodman HM, Boyer HW. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J Virol 1974; 14:1235-1244
  • Huang Q, Baum L, Fu WL. Simple and practical staining of DNA with GelRed in agarose gel electrophoresis. Clinical Laboratory 2010; 56:149-152
  • Huang Q, Fu WL. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clin Chem Lab Med 2005; 43:841-842
  • Johnson PH, Grossman LI. Electrophoresis of DNA in agarose gels. Optimizing separations of conformational isomers of double- and single-stranded DNAs. Biochemistry 1977; 16:4217-4225
  • Lai E, Birren BW, Clark SM, Simon MI, Hood L. Pulsed field gel electrophoresis. Biotechniques 1989; 7:34-42
  • Lee PY, Costumbrado J, Hsu C-Y, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments 2012; 3923
  • Lee SV, Bahaman AR (2012) Discriminatory power of agarose gel electrophoresis in DNA fragments analysis. In: Magdeldin S (ed) Gel Electrophoresis Principles and Basics. IntechOpen, pp 41-55
  • Li C, Arakawa T. Agarose native gel electrophoresis of proteins. Int J Biol Macromol 2019; 140:668-671
  • Mathew MK, Smith CL, Cantor CR. High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature. Biochemistry 1988; 27:9204-9210
  • Miller SE, Taillon-Miller P, Kwok PY. Cost-effective staining of DNA with SYBR green in preparative agarose gel electrophoresis. Biotechniques 1999; 27:34-36
  • Moore D, Dowhan D. Purification and Concentration of DNA from Aqueous Solutions. Curr Protoc Mol Biol 2002; 59:2.1.1-2.1.10
  • Philippsen P, Zachau G. Partial degradation of transfer RNAs and transfer RNA fragments by spleen phosphodiesterase as studied by disc electrophoretic methods. Biochim Biophys Acta 1972; 277:523-538
  • R Development Core Team (2017) R: A Language and Environment for Statistical Computing In. R Foundation for Statistical Computing, Austria, Vienna
  • Ross PD. Electrophoresis of DNA. I. On a relationship between electrophoresis and donnan equilibrium experiments on DNA. Biopolymers 1964; 2:9-14
  • Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, USA
  • Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 2012; 9:671-675
  • Serwer P. Agarose gels: Properties and use for electrophoresis. Electrophoresis 1983; 4:375-382
  • Sharp PA, Sugden B, Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry 1973; 12:3055-3063
  • Sigmon J, Larcom LL. The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis. Electrophoresis 1996; 17:1524-1527
  • Slater GW, Noolandi J. The biased reptation model of DNA gel electrophoresis: mobility vs molecular size and gel concentration. Biopolymers 1989; 28:1781-1791
  • Smith DR. Agarose gel electrophoresis. Methods Mol Biol 1993; 18:433-438
  • Stellwagen NC. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis 2009; 30 Suppl 1:188-195
  • Thorne HV. Electrophoretic characterization and fractionation of polyoma virus DNA. J Mol Biol 1967; 24:203-211

Effect of DNA Concentration on Band Intensity and Resolution in Agarose Gel Elec-trophoresis

Yıl 2021, Cilt: 14 Sayı: 3, 326 - 333, 30.12.2021
https://doi.org/10.52976/vansaglik.969547

Öz

Objective: Agarose gel electrophoresis (AGE) is a widely used method for separating, identifying, and purifying nucleic acids. It has been used intensively in molecular researches and biotechnological applications. In AGE, band resolution and band quality are important parameters for researchers. Until now, many factors such as comb thickness, gel concentration, voltage and buffers have been reported to influence resolution and band quality in AGE. However, effect of DNA concentration on band resolution and intensity in AGE is unclear. Therefore, aim of the study was to investigate the effect of DNA concentration on AGE results in detail.
Material and Method: Different concentrations of DNA Marker and specific DNA fragments obtained by PCR were analyzed, but the loaded total DNA quantity was not changed. Furthermore, the effect of DNA concentra-tion on AGE was also investigated by designed gel combs with different thicknesses and wideness.
Results: It was shown that DNA concentration did not affect gel resolution, and the effectwas not changed by comb thickness and wideness. Also, 2-fold dilution did not affect band intensities while 8-fold dilution signifi-cantly affected band intensities of all tested DNA fragments.
Conclusion: Concentrating samples, at least 8-fold, and using thin and narrow gel combs can be used for de-sired results in AGE.

Kaynakça

  • Aaij C, Borst P. The gel electrophoresis of DNA. Biochim Biophys Acta 1972; 269:192-200
  • Arslan M. A new primer designing for PCR-RFLP analysis of A and B genetic variants of bovine kappa-casein. Harran Üniversitesi Veteriner Fakültesi Dergisi 2020; 9:6-11
  • Bachvaroff R, McMaster PR. Separation of microsomal RNA into five bands during agar electrophoresis. Science 1964; 143:1177-1179
  • Greaser ML, Warren CM. Protein electrophoresis in agarose gels for separating high molecular weight proteins. Methods Mol Biol 2012; 869:111-118
  • Green MR, Sambrook J. Analysis of DNA by Agarose Gel Electrophoresis. Cold Spring Harbor Protocols 2019; 2019
  • Hall AC. A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis. bioRxiv 2020; 568253
  • Helling RB, Goodman HM, Boyer HW. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J Virol 1974; 14:1235-1244
  • Huang Q, Baum L, Fu WL. Simple and practical staining of DNA with GelRed in agarose gel electrophoresis. Clinical Laboratory 2010; 56:149-152
  • Huang Q, Fu WL. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clin Chem Lab Med 2005; 43:841-842
  • Johnson PH, Grossman LI. Electrophoresis of DNA in agarose gels. Optimizing separations of conformational isomers of double- and single-stranded DNAs. Biochemistry 1977; 16:4217-4225
  • Lai E, Birren BW, Clark SM, Simon MI, Hood L. Pulsed field gel electrophoresis. Biotechniques 1989; 7:34-42
  • Lee PY, Costumbrado J, Hsu C-Y, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments 2012; 3923
  • Lee SV, Bahaman AR (2012) Discriminatory power of agarose gel electrophoresis in DNA fragments analysis. In: Magdeldin S (ed) Gel Electrophoresis Principles and Basics. IntechOpen, pp 41-55
  • Li C, Arakawa T. Agarose native gel electrophoresis of proteins. Int J Biol Macromol 2019; 140:668-671
  • Mathew MK, Smith CL, Cantor CR. High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature. Biochemistry 1988; 27:9204-9210
  • Miller SE, Taillon-Miller P, Kwok PY. Cost-effective staining of DNA with SYBR green in preparative agarose gel electrophoresis. Biotechniques 1999; 27:34-36
  • Moore D, Dowhan D. Purification and Concentration of DNA from Aqueous Solutions. Curr Protoc Mol Biol 2002; 59:2.1.1-2.1.10
  • Philippsen P, Zachau G. Partial degradation of transfer RNAs and transfer RNA fragments by spleen phosphodiesterase as studied by disc electrophoretic methods. Biochim Biophys Acta 1972; 277:523-538
  • R Development Core Team (2017) R: A Language and Environment for Statistical Computing In. R Foundation for Statistical Computing, Austria, Vienna
  • Ross PD. Electrophoresis of DNA. I. On a relationship between electrophoresis and donnan equilibrium experiments on DNA. Biopolymers 1964; 2:9-14
  • Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, USA
  • Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 2012; 9:671-675
  • Serwer P. Agarose gels: Properties and use for electrophoresis. Electrophoresis 1983; 4:375-382
  • Sharp PA, Sugden B, Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry 1973; 12:3055-3063
  • Sigmon J, Larcom LL. The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis. Electrophoresis 1996; 17:1524-1527
  • Slater GW, Noolandi J. The biased reptation model of DNA gel electrophoresis: mobility vs molecular size and gel concentration. Biopolymers 1989; 28:1781-1791
  • Smith DR. Agarose gel electrophoresis. Methods Mol Biol 1993; 18:433-438
  • Stellwagen NC. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis 2009; 30 Suppl 1:188-195
  • Thorne HV. Electrophoretic characterization and fractionation of polyoma virus DNA. J Mol Biol 1967; 24:203-211
Toplam 29 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Veteriner Cerrahi
Bölüm Orijinal Araştırma Makaleleri
Yazarlar

Mevlut Arslan 0000-0003-4883-4736

Erdem Tezcan 0000-0001-7379-9931

Haluk Camcı 0000-0003-4763-8696

Murat Kemal Avcı 0000-0001-5211-2352

Yayımlanma Tarihi 30 Aralık 2021
Gönderilme Tarihi 10 Temmuz 2021
Yayımlandığı Sayı Yıl 2021 Cilt: 14 Sayı: 3

Kaynak Göster

APA Arslan, M., Tezcan, E., Camcı, H., Avcı, M. K. (2021). Effect of DNA Concentration on Band Intensity and Resolution in Agarose Gel Elec-trophoresis. Van Sağlık Bilimleri Dergisi, 14(3), 326-333. https://doi.org/10.52976/vansaglik.969547

ISSN 

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